Regulation of the type IV pili localization in Myxococcus xanthus

Myxococcus xanthus cells are rod-shaped and move in the direction of their long axis, using two distinct motility systems. The S-motility system is type IV pili (T4P)-dependent. T4P are dynamic structures, localized at the leading cell pole and undergo extension/retraction oscillations. Upon retract...

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Bibliographic Details
Main Author: Bulyha, Iryna
Contributors: Sogaard-Andersen, L. (Thesis advisor)
Format: Doctoral Thesis
Language:English
Published: Philipps-Universität Marburg 2010
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Summary:Myxococcus xanthus cells are rod-shaped and move in the direction of their long axis, using two distinct motility systems. The S-motility system is type IV pili (T4P)-dependent. T4P are dynamic structures, localized at the leading cell pole and undergo extension/retraction oscillations. Upon retraction T4P generate a mechanical force, large enough to pull a cell forward. Regulation of T4P extension/retraction dynamics relies on two motor proteins, PilB and PilT, which are members of the superfamily of secretion ATPases. PilT is the only protein required for retraction. Genetic and biochemical analyses suggest that PilB and PilT function antagonistically and that ATP hydrolysis by PilB provides the energy for T4P extension, while the energy for T4P retraction is provided by ATP hydrolysis by PilT. How the activities of PilB and PilT are regulated to provide temporal separation of T4P extension and retraction is not known. Although several models have been proposed, it is still not clear how mechanical force is generated in the second motility system, the A-motility system. As M. xanthus cells move over a surface, they occasionally stop and then resume gliding in opposite direction, with the old lagging pole becoming the new leading pole and vice versa. The Frz chemosensory system regulates the reversal frequency. Importantly, during reversals the two motility systems change their polarity synchronously. To investigate the molecular mechanisms underlying T4P extension/retraction and T4P pole-to-pole oscillations during a reversal, the cellular localization of six conserved T4P proteins (PilB, PilT, PilQ, PilC, PilN and PilM) was determined. These six proteins in combination localize to three different subcellular compartments – the outer membrane, inner memrane and cytoplasm. We found that PilB, PilT, PilQ, PilC, PilN and PilM localized in three distinct polar patterns. The outer membrane secretin PilQ, the inner membrane proteins PilC and PilN and the MreB/FtsA-like cytoplasmic protein PilM localized to both poles in a symmetric pattern. Notably, this pattern did not change during reversals. Moreover, no differences in the localization of PilQ, PilC, PilN and PilM were observed in the absence of an active Frz system. Thus, we propose that PilQ, PilC, PilN and PilM are stationary T4P components, which do not oscillate from pole to pole during cellular reversal. Furthermore, we found that the cytoplasmic proteins PilB and PilT localized to the opposite poles. PilB, the extension motor, localized predominantly at the piliated cell pole, whereas PilT, the retraction motor, predominantly at the non-piliated cell pole. Using time-lapse microscopy, we directly observed pole-to-pole relocation of YFP-PilT during cellular reversals, which did not occur in the absence of the Frz system. We also observed clear differences in the PilB localization in the WT and in a frz mutant. In WT, three distinct PilB localization patterns were observed in immunofluorescence microscopy with anti-PilB antibodies: unipolar (40% of cells), bipolar asymmetric (35%) and bipolar symmetric (25%). In a frz mutant, however, the ratio shifted towards bipolar symmetric localization. We conclude that the molecular motors PilB and PilT are dynamic T4P components and oscillate between poles during reversals. Hence, T4P pole-to-pole oscillations in M. xanthus involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. In addition, YFP-PilT displayed noisy accumulation at the piliated pole between reversals, and FRAP experiments revealed rapid turnover of YFP-PilT in the polar clusters between reversals. Taken together, these observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. The Frz system regulates the dynamic localization of PilB and PilT during reversals. In addition, we found that the Ras-like GTPase MglA and its paralog SofG regulate the correct polarity of PilB and PilT. Specifically, we found that MglA is a nucleotide-dependent molecular switch that establishes correct PilT polarity and regulates its dynamic localization during reversals. SofG is required for establishing the correct localization/polarity of PilB and PilT and also inhibits T4P assembly at the lagging cell pole.
DOI:10.17192/z2010.0453